NANOGP8 Function in Stemness: A major question in our research is why the retrogene NANOGP8 located on Chromosome 15 is activated in human carcinomas and leukemias. Its coding region differs from NANOG?s located on chromosome 12 by 5 nucleotides that cause only 2 amino acid changes in a protein of 305 amino acids. It has not been clear that NANOGP8 could rescue stemness if NANOG is lost. This year we have found that when shRNA inhibits the expression of both NANOG and NANOGP8 and as a consequence the ability of human colorectal carcinoma cells (CRC) to form spheroids, the re-expression of NANOGP8 rescues the ability of CRC lines to form spheroids whereas re-expression of NANOG rescues only one of the cell lines. Since the capacity of cells to form spheroids is a major in vitro measure of stemness, the demonstration that NANOGP8 rescues stemness in these carcinomas suggests that NANOGP8 is able to replace NANOG as a core transcription factor. These data are in a manuscript undergoing final review at the journal Oncogene. Mechanism of Apoptosis: A goal for this year under aim 2 was to elucidate whether allele-specific shRNA inhibition of NANOG or NANOGP8 induces apoptosis. Lentiviral vector delivered shRNA to either NANOG causes the appearance of phosphatidylserine, a marker for apoptosis, in monolayer culture in 3 human colorectal carcinoma cells. However, since NANOG or NANOGP8 expression is low in monolayer culture, the amount of apoptosis is low in monolayer cultures. When human colorectal carcinoma cells that normally grow attached to a substrate in vitro are placed in suspension culture without an ability to attach to a substrate, they die from a form of apoptosis termed anoikis that is driven mainly by the activation of Caspase 8 and the extrinsic pathway of apoptosis. Since the relative expression of both NANOGs increases in suspension culture, we investigated whether inhibition of either NANOG caused apoptosis and if so what pathway for apoptosis. We found that inhibition of NANOGP8 and to a lesser extent NANOG induces apoptosis through the intrinsic pathway by activation of Caspases 9 and 3 to reduce tumor growth. Confirmation of this observation was provided by stimulating growth of CRC cells in suspension culture by blocking the effects of allele-specific shRNAs by treatment with inhibitors of Caspases 3 and 9 as well as the overexpression of NANOGP8 and NANOG. These data suggest that the induction of apoptosis may be a useful component for therapy. Synergy with Chemotherapy: The interaction between inhibition of the NANOGs and a model of chemotherapy has been addressed. Since camptothecins are important agents for the clinical management of CRC, we have assessed whether the effects of Topotocan, a model camptothecin, might be synergistic with lentiviral vector delivered shRNA. Topotecan is a Topoisomerase I inhibitor that induces DNA damage that in turn causes cytotoxicity through stimulation of both the extrinsic and intrinsic pathways of apoptosis. Topotecan induces cell death in a dose dependent fashion and with consistent kinetics that will generally achieve a &gt; 90% kill in CRC lines sensitive to topotecan in 96 hr. Interestingly, relative transcript levels of both NANOGs increase at 72 hr after topotecan exposure. When lentiviral allele-specific shRNAs are added to CRC lines in monolayer culture along with topotecan, shRNA inhibition of NANOGP8 led to greater reduction in all 3 CRC lines than with Topotecan alone. Both the intrinsic and extrinsic pathways of apoptosis were stimulated. Further, the caspase inhibitors at the concentrations that blocked anoikis did not reverse the inhibition of growth caused by topotecan and shRNAs. This suggests that lentiviral shRNA to NANOG or NANOGP8 may be synergistic with topotecan treatment. Regulation of NEDD9: While it is possible to link inhibition of NANOGP8 or NANOG with the induction of apoptosis under conditions in which there is apoptotic stress, it is not clear what molecules link inhibition of either NANOG with the induction of apoptosis. To assess this, RNA-seq has been done in 2 CRC lines cultured in monolayer and suspension as well as with shRNA to NANOGP8 or with overexpression with NANOGP8. Ingenuity Pathway Analysis of the genes modulated by alterations in NANOGP8 identified NEDD9 as a critical gene. NEDD9 is a scaffolding protein for src and FAK in adhesion plaques that activates AKT and is an anti-apoptotic stimulus since it contributes to constitutive activation of FAK. RT-PCR confirmed that NEDD9 expression was inhibited by shRNA to the NANOGs and increased by overexpression of either NANOG in the CRC lines tested. In addition, ChIP assays confirmed that NANOG binds the promoter of NEDD9. Subsequent research is assessing whether this is the mechanism by which apoptosis is activated. We are currently assessing the role of NANOG and NEDD9 as prognostic factors in a series of nearly 400 primary colon carcinomas. Finally, the human DNA binding site for human NANOG has not been identified and we intend to define that site within this promoter. We have identified a minimal region of the promoter that responds to NANOG and in the coming year we will further narrow the binding site. These results are currently being put into a manuscript that will be submitted this year. Transduction in Vivo: About 6 months ago we started a DOD grant that will assess the potential of allele-specific lentiviral vector delivered shRNA as a gene therapy to inhibit growth of established tumor in preclinical xenograft models. Initial experiments indicated that intratumoral injection of lentiviral shRNA induced a modest nonspecific inhibition of tumor growth. Since the inhibition was as great to the negative control vector as the specific shRNAs, the lentiviral constructs induced an innate immune response. Repeated assessment of intratumoral injections with the lentiviral shRNA failed to reveal any significant transduction of tumor cells within the subcutaneous tumor nodules as measured by the presence of Green Fluorescent Protein (GFP) fluorescence. GFP is included in all of our lentiviral shRNA constructs as a reporter and is present in 45- 80% of cells within spheroids that are transduced in vitro. There was no GFP expression within tumor cells 3 ? 5 days after intratumoral injection into small (3 mm diameter) nodules. We postulate that there may be 4 reasons why this may occur: 1) the amount of virus injected was too low for transduction, 2) interstitial pressure prevents binding of standard pseudotyped lentiviral particles to tumor cells, 3) intratumoral acidity decreases viral binding and fusion to tumor cell membranes so that transduction is blocked and 4) host stroma inhibits binding of vector to tumor cells. To assess postulate 1) we have developed a collaboration with Dr. J. Reiser at FDA/CBER who produces high quality/high titer lentivirus for selective targeting and use in clinical situations. To assess postulates 2 and 3 we have developed a collaboration with Dr. Breckpot in Belgium to develop a better and more selective binding agent that may overcome interstitial pressure and acidity. Associate Professor Karine Breckpot at the Brussels Vrje Universtat is a leading authority on innate immunity to lentivirus. She has developed a natural single chain immunoglobulin from llamas that recognizes CEA. She has given us this targeting plasmid that we are just beginning to incorporate into a lentiviral vector and with her and Dr. Reiser's guidance we are beginning to test viral binding and specificity in vitro before ramping up production for preclinical testing. To assess postulate 4) we will do co-cultures of mouse 3T3 cells with human CRC cells to assess whether host stroma also may participate in inhibition of lentiviral transduction.